Integrative Functional Assays, Chemical Genomics and High Throughput Screening: Harnessing Signal Transduction Pathways to a Common HTS Readout

Abstract

Chemical genomics is a drug discovery strategy that relies heavily on high-throughput screening (HTS) and therefore benefits from functional assay platforms that allow HTS against all relevant genomic targets. Receptor Selection and Amplification Technology (R-SAT) is a cell-based, high-throughput functional assay where the receptor stimulus is translated into a measurable cellular response through an extensive signaling cascade occurring over several days. The large biological and chronological separation of stimulus from response provides numerous opportunities for enabling assays and increasing assay sensitivity. Here we review strategies for building homogeneous assay platforms across large gene families by redirecting and/or amplifying signal transduction pathways.

Identification of the First Synthetic Steroidogenic Factor 1 Inverse Agonists: Pharmacological Modulation of Steroidogenic Enzymes

“…R-SAT defines a broadly applicable cell proliferation assay that allows for the pharmacological study of various receptor targets and their ligands, including G protein-coupled receptors, cytokine receptors, and nuclear hormone receptors (Piu et al, 2002;Burstein et al, 2006;Piu et al, 2006). As expected, SF-1 was highly constitutively active in this assay ( Fig.…”

“…Receptor Selection and Amplification Technology is a functional cell-based assay that allows one to monitor receptordependent proliferative responses and has been described elsewhere (Piu et al, 2002). The technology has been validated for a number of receptors including G protein-coupled receptors, receptor tyrosine kinases, cytokine receptors, and nuclear receptors (Piu et al, 2002Burstein et al, 2006). Its principle resides in the genetic selection and amplification of the nuclear receptors in a liganddependent manner.…”

Identification of the Antiarrhythmic Drugs Amiodarone and Lorcainide as Potent H3 Histamine Receptor Inverse Agonists

“…We developed a high-throughput cellular proliferation assay that is compatible with most GPCRs and that detects constitutive receptor activity with high sensitivity (R-SAT) (Burstein et al, 2006). To identify novel small molecule ligands, R-SAT was used to screen the human H3 histamine receptor against a collection of more than 200,000 compounds.…”

“…Cells were transiently transfected with 1-10 ng/well receptor DNA, and 30 ng/well pSI-b-galactosidase (Promega Corporation) per well of a 96-well plate using Polyfect (Qiagen, Valencia, CA) according to the manufacturer's instructions. In addition, "helper" DNAs encoding accessory proteins were transfected including 20 ng/well ras/rap1B(AA), 2 ng/well adenylyl cyclase 2, 4 ng/ well Gqi5 (for H3 and H4 receptors only), 0.8 ng/well Grk2, and 6 ng/ well RGS1 (Burstein et al, 2006(Burstein et al, , 2011. One day after transfection, media were changed, and cells were combined with ligands in DMEM supplemented with 25% Ultraculture synthetic supplement (Cambrex, Walkersville, MD) instead of calf serum to a final volume of 200 ml/well.…”

“…Using a cellular proliferation assay called receptor selection and amplification technology (R-SAT) (Burstein et al, 2006), we identified lorcainide and amiodarone from a diverse collection of antiarrhythmic drugs as potent H3 histamine receptor antagonists. Further profiling revealed that both agents display inverse agonist activity at certain H3 receptor isoforms, as well as surprising selectivity for H3 receptors over other GPCRs.…”